Activity of antibiotics against extracellular and intracellular forms of Staphylococcus aureus

Maritza Barcia-Macay (Patterson) Unité de Pharmacologie cellulaire et moléculaire Activity of antibiotics against extracellular and intracellular forms of Staphylococcus aureus Pharmacodynamic studies in vitro using a model of human THP-1 macrophages

I. INTRODUCTION

Staphylococcus aureus Bacteria with the greatest pathogenic potential in human infections. A major cause of nosocomial and community-acquired infections.

S. aureus infections 1. skin and soft tissue infections impetigo erysipelas cellulitis furuncle abscess

2. food-poisoning S. aureus infections

S. aureus infections 3. Deep-organ infections endocarditis pneumonia osteomyelitis

S. aureus infections All these infections are difficult to treat : recurrence / persistence in relation with the intracellular character of S. aureus selection of antibiotics based on pharmacokinetic / pharmacodynamics properties resistance to currently available antibiotics need of drugs acting on multiresistant strains

Intracellular infection by S. aureus Normal process of phagocytosis 1) attachment to phagocyte membrane 2) ingestion (phagosome) 3) fusion lysosome and phagosome 4) bacterial destruction 5) digestion and release of microbial debris

Intracellular infection by S. aureus Incomplete phagocytosis of S. aureus 1) attachment to phagocyte membrane 2) ingestion (phagosome) 3) fusion lysosome and phagosome X 4) bacterial destruction X 5) digestion and release of microbial debris

Intracellular infection by S. aureus Bacteria able to survive in different host cells : phagocytes and non-phagocytes Neutrophils Macrophages Mammary epithelial cells Enterocytes Keratinocytes Osteoblasts Fibroblasts Endothelial cells Brouillette et al, Vet Microbiol (2004) 101:253-262; Microb Pathog. (2003) 35:159-68.

Antibiotic pharmacokinetics / pharmacodynamics general concept Dosage regimen Concentration versus time in serum absorption distribution elimination Concentration versus time in tissues and other body fluids Concentration versus time at site of infection Pharmacologic or toxicologic effect Antimicrobial effect versus time PHARMACOKINETICS PHARMACODYNAMICS Craig CID (1998) 26:1-10

Antibiotic pharmacokinetics / pharmacodynamics intracellularly PHARMACOKINETICS Carryn et al, Infect Dis Clin North Am. (2003) 17:615-34 PHARMACODYNAMICS

S. aureus resistance The world first commercially available antibiotic appeared in 1941 1941 1960 YEAR OF REPORTED RESISTANCE 2000 Introduction of Penicillin Aminoglycosides, 1950 Oxazolidinones Macrolides, 1952 Glycopeptides, 1956 Methicillin, 1960 Quinolones, Streptogramins, 1962

S. aureus resistance Most worrying resistance phenotypes having emerged over time year phenotype first description 1960 HA-MRSA England 1967 MDR HA-MRSA Europe, Australia, Japan 1980 Genta-R MRSA USA, Ireland, UK 1993 CA-MRSA Australia 1997 VISA Japan 2002 VRSA USA Grundmann et al., Lancet (2006) 368:874-85

S. aureus resistance Percentage of MRSA resistance in Europe in 2004: S. aureus proportion of invasive isolates MRSA in 2004 Data from the European antimicrobial resistant surveillance system, EARSS.

S. aureus resistance Prevalence of resistance to other antibiotic classes MSSA MRSA 100 80 60 40 20 % resistance amoxi amoxi-clav glycopeptides macrolides lincosamides synergistins aminoglycosides tetracyclines rifampicin fluoroquinolones 0 Fluit et al., J Clin Microbiol (2001) 39: 3727-3732

Need for new antistaphylococcal agents A lot of drugs in the pipeline recent and novel agents for S. aureus recently brought on the Belgian market on the market; not yet in Belgium (late) stage of clinical development investigational moxifloxacin linezolid synercid daptomycin tigecycline telavancin oritavancin dalbavancin CS-023/PZ-601 MX-2401 API-1252 ceftobiprole DK-619 iclaprim retapamulin WCK-771 new oxazolidinones new ketolides... What will be your choice? Sympo S. aureus 11/01/07-8 F. Van Bambeke, symposium on S. aureus Brussels, 11-1-2007

II. AIM OF THE STUDY

recurrence / persistence in relation with the intracellular character of S. aureus selection of antibiotics based on pharmacokinetic / pharmacodynamics properties To develop an intracellular model allowing to compare the activity of antibiotics on a pharmacodynamic basis resistance to currently available antibiotics need of drugs acting on multiresistant strains To evaluate the cellular pharmacokinetics and the intracellular activity towards multiresistant strains of a new antibiotic in development

III. METHODOLOGY

Setting-up of the intracellular model Method 1) opsonization of S. aureus with human serum 2) phagocytosis of the bacteria by THP-1 macrophages (ratio 4 bacteria vs 1 macrophage) 3) elimination of extracellular S. aureus (gentamicin 100 X MIC). Rinse of infected macrophages (time-zero) 4) intracellularly infected macrophages, ready to test antibiotic activity 5) maintenance of gentamicin at its MIC during the whole incubation period for controls to avoid extracellular contamination

Setting-up of the intracellular model cell line? THP-1= many features of human monocytes/macrophages parameter Tsuchiya, Int. J. Cancer (1980) 26:171-176; Auwerx, Experientia (1991) 47:22-31 morphological features cytochemical features surface antigens and receptors secreted proteins functional features characteristics morphology resembling that of monocytic leukemia cells: - diameter 12-14 µm - moderate basophile cytoplasm - small azurophiles granules, few vacuoles - nuclei irregular in shape - positive for α-naphthyl butyrate esterase - negative reaction with periodic acid-schiff and Sudan black B - diploid (46,XY) chromosome number CD4, CD30, Factor X receptor, Factor Xa receptor, FcRI, FcRII, GM-CSF-receptor, HDL receptor, LDL receptor, TNF receptor, C3b receptor, LFA-1 receptor, Fibronectin receptor, Leu M1, Leu M2, Leu M3, HLA-DR antigens, scavengers receptors - hormones, cytokines: TNF-α, IL-1, IL-1b, CSF-1, M-CSF, erythrocyte differentiation factor, PDGF-1 and 2, thymosin B4, killer T cell activating factor, monocyte chemotactic factor - enzymes: lipoprotein lipase, lyzozyme - binding proteins: apoprotein E - phagocytosis - production of lyzozyme - capacity to restore the lympocyte T mitogenic responsiveness

Setting-up of the intracellular model bacterial strain? ATCC 25923 clinical isolate from 1976 fully susceptible widely used as a standard for microbiological testing of antibiotics useful to compare all antibiotics towards a single strain, but may differ in virulence with current strains

Setting-up of the intracellular model antibiotics? Beta-lactams: first choice for susceptible strains Aminoglycosides: highly bactericidal extracellularly Rifampicin: considered as first choice for intracellular infections Macrolides, quinolones: high cellular accumulation Glycopeptides: alternative for MRSA Linezolid: recently introduced, active on MRSA

S. aureus intracellular model gentamicin prevents extracellular contamination Bacteria multiply in phagolysosomes where ph is acidic membrane bond vacuole

General protocol Assessment of antibiotic extracellular and intracellular activity EXTRACELLULAR INTRACELLULAR 4 bacteria/cell 10 6 CFU/ml 10 6 CFU/mg protein Incubation over 24 h with antibiotics Colony counting (CFU/ml) Collection and lysis of cells (sonication) CFU/protein content Antibiotic concentration (microbiological, radiochemical, fluorimetric assays)

IV. RESULTS

First goal of this thesis Human macrophages Fully susceptible strain

Antibiotics accumulate to variable levels in THP-1 macrophages High level: macrolides oritavancin rifampin moderate level: aminoglycoside old glycopeptides quinolones low level: linezolid β-lactams

influence of time on activity low to moderate accumulation moderate to high accumulation GEN, RIF, ORI quickly cidal OXA, MXF, ORI slowly cidal in all cases, intracellular activity << extracellular activity no direct relation between accumulation and intracellular activity

concentration-effect relationships sigmoidal relationships

concentration-effect relationships Emax Emax Emax Emax sigmoidal relationships Emax intra << Emax extra

concentration-effect relationships EC50 EC50 EC50 EC50 sigmoidal relationships Emax intra << Emax extra EC 50 extra = EC 50 intra for OXA and MXF EC 50 extra < EC 50 intra for GEN and ORI

intracellular killing is visible! control OXA ORI

extracellular versus intracellular activity growth killing -5 intracellular Δ log CFU from time 0-4 -3-2 -1 0 0 AZM -1 LNZ TEL -2 RIFCIP AMP TEC VAN NAF PEN V GEN -3 ORI GRN MXF LVX TLV OXA -4-5 killing growth Poorly active against extra and intra S. aureus extracellular Δ log CFU from time 0

extracellular versus intracellular activity growth killing -5 intracellular Δ log CFU from time 0-4 -3-2 -1 0 AZM LNZ TEL ORI GRN MXF LVX TLV OXA RIFCIP AMP TEC VAN NAF PEN V GEN killing growth Active against extra S. aureus 0-1 -2-3 -4-5 extracellular Δ log CFU from time 0

extracellular versus intracellular activity growth killing intracellular Δ log CFU from time 0-5 -4-3 -2-1 0 AZM LNZ TEL ORI GRN MXF LVX TLV OXA RIFCIP AMP TEC VAN NAF PEN V GEN killing growth Best choice for activity against extra and intra S. aureus 0-1 -2-3 -4-5 extracellular Δ log CFU from time 0

conclusion model of infection of human macrophages by S. aureus over 24 h allowing for the study of influence of time and concentration on antibiotic activity relation between activity and accumulation intracellular activity << extracellular activity no correlation with level of accumulation impairing effect of acidic ph on some antibiotics optimizing antibiotic efficacy choice of the drug (active extra and intracellularly) optimization of exposure ( time and concentration)

Second goal of this thesis New drug in development Different phenotypes of resistance

Telavancin, a new glycopeptide Hemi-synthetic derivative of vancomycin, with new mode of action and new pharmacokinetic profile permeabilization of bacterial membrane prolonged half-life dimerization and cooperative binding to peptidoglycan precursors shortening of half-life

MIC and MBC against S. aureus with different resistance phenotypes MIC and MBC of vancomycin and telavancin against the S. aureus strains used. vancomycin telavancin phenotype strain MIC MBC MIC MBC MSSA ATCC25923 1 1 0.5 0.5 ATCC29213 1 1 0.5 0.5 MRSA ATCC33591 2 4 0.5 1 ATCC43300 2 2 0.5 0.5 VISA NRS23 4 4 0.5 0.5 NRS52 4 4 0.5 0.5 VRSA VRS1 >128 >256 4 8 VRS2 16 64 2 8 more active than VAN against VISA and VRSA bactericidal against all strains

Influence of time on EXTRACELLULAR activity VAN vs TLV MSSA ATCC 25923 Telavancin is more rapidly cidal than vancomycin

Influence of concentration on EXTRACELLULAR activity VAN vs TLV MSSA ATCC 25923 at 3 h, TEL shows bimodal conc-dependent effects at 24 h, both drugs are bactericidal at high concentrations

EXTRACELLULAR activity of telavancin: comparison of different strains TLV versus MSSA, MRSA, VISA, VRSA at 3 h, TEL shows bimodal conc-dependent effects towards MSSA and MRSA

INTRACELLULAR activity of vancomycin and telavancin towards different strains VAN and TLV versus MSSA, MRSA, VISA, VRSA TEL shows bimodal conc-dependent effects towards MSSA / MRSA VAN is only static intracellulalry

Why bimodal effects for telavancin? VAN and TLV: inhibition of peptidoglycan synthesis In MSSA and MRSA, telavancin can exert multiple modes of action Δ log CFU from time 0 3 2 1 0-1 -2-3 -4-5 vancomycin telavancin -2-1 0 1 2 3 log concentration (µg/ml) TLV: membrane permeabilization Higgins et al., AAC (2004) 49:1127-34

Telavancin cellular pharmacokinetic data rationalizing its intracellular activity (studies with J774 macrophages)

subcellular distribution of telavancin Same distribution as a lysosomal enzyme High concentration in the compartment where S. aureus sojourns!

conclusion model of infection of human macrophages by S. aureus over 24 h applicable to multiresistant strains vancomycin slowly bactericidal extracellularly (MSSA and MRSA) poorly or not active on VISA and VRSA static intracellularly telavancin bactericidal extra- and intracellularly, including against resistant strains bimodal effect against MSSA and MRSA could be related to multiple modes of action high accumulation in the infected compartment

V. GENERAL CONCLUSION: can we do better?

Limitations of the model and perspectives for future work Constant concentrations (pharmacokinetic variations not taken into account): develop dynamic models Protein binding (free fraction is active and able to accumulate) develop in vivo models Phagocytic cells (S. aureus also infects non phagocytic cells where its fate may be different) develop models of infection in non-phagocytic cells Testing of antibiotics alone (combinations often used in the clinics to cope with resistance) testing of drug combinations

THANK YOU!

THANKS TO : Prof. M.-P. Mingeot-Leclercq Dr. C. Seral (Spain) Prof. J.- P. Herveg Prof. V. Préat Prof. M. Delmée Prof. Y. Glupczynski Prof. B. Gallez Prof. A. Pascual (Spain) Prof. M. Struelens (ULB) Theravance (USA)

HURRA FACM!!! Special thanks to O. Meert and M.-C. Cambier

THANKS TO YOU ALL